Isolation And Expression Analysis Of The NTLEAFY And NTA Gene In Narcissus Tazetta Var. Chinensis Roem

Narcissus tazetta var.chinensis Roem,an ornamental plant of the family Amaryllidaceae that grows from a bulb and bloom during the Chinese Spring Festival,and it is a famous traditional Chinese flower with high economic and ornamental value.As a homologous triploid plant,it is difficult to create desirable varieties by traditional hybridization,and the wild Narcissus resources are extremely deficient in China.Narcissus is a perennial plant with high temperatures requirement for floral buds development while bloom at lower temperatures.In addition,the Narcissus is affected by disease and insect pest severely,so it is confronted with more pressure and severe challenges in worldwide flowers industry.In this study,gene NTLEAFY involved in floral organ development and lectin gene NTA were isolated by RT-PCR approach,its nucleotide and amino acid sequences were analyzed.And the genetic transformation system was established for functional study.The major finding is as follows:1.A full-length cDNA of a LEAFY homologues NTLEAFY was cloned from Chinese Narcissus. With a size of 1340 bp,the cDNA contains an 1137 bp open reading frame(ORF) encoding a putative protein of 378 amino acids.The pI and molecular weight of the protein NTLEAFY were predicted to be 8.00 and 42274.65 Da,respectively.Homologous analysis showed that the sequence similarity between NTLEAFY and LEAFY homologues from species as Narcissus tazetta,Hyacinthus orientalis,et al.is over 76%.Comparison of the predicted amino acid sequence of NTLEAFY to LFY homologues from several other plant species such as Narcissus tazetta,Hyacinthus orientalis,Arabidopsis thaliana et al. reveals an overall homology at the amino acid level of over 57%and a similar pattern of variable regions and conserved in C-terminal.The prediction of the protein structure indicated that tertiary protein structure of NTLEAFY from Chinese Narcissus is similar to that of Arabidopsis thaliana.Real-time PCR was used to analyse the expression of NTLEAFY in different organs and stages of development and the data were normalized in relation to housekeeping gene GAPDH.It was found that the narcissus NTLEAFY gene was expressed not only in reproductive organs,but also in vegetative organs.The results revealed that the transcript levels of NTLEAFY was high in flower buds,but only 1/8 of the amount was detected in the fully blooming flowers.Within blooming flower,transcript levels was higher in stamens than in pistil,perianth and corona-crown indicating that NTLEAFY gene is important both in vegetative growth and reproductive growth.Wild-type Arabidopsis plants were transformed by the floral dip method,and transgenic plants were obtained.However,early blooming has not been observed in NTLEAFY transgenic lines as expected and the transgenic plants were small and with abnormal leaves.In shoot apex of the transgenic plant,leaves and inflorescence exhibit the shape of flowers,and flowers with 5 petals were observed.The result indicated that NTLEAFY plays an essential role in the regulation of blooming time and floral organ identity.2.A full-length cDNA of a lectin gene designated as NTA was cloned from Chinese Narcissus with the method of RT-PCR.With a size of 698 bp including a 519 bp open reading frame,the NTA encodes a precursor lectin with 172 amino acids.The pI and molecular weight of the protein NTA were predicted to be 5.84 and 18 615.19 Da,respectively.The homologous analysis showed that the sequence similarity between NTA and lectin gene from other monocotyledon species were over 82%,respectively.The predicted amino acid sequence of NTA to lectin protein from other monocotyledon species were over 77%,respectively.The prediction of the protein structure indicated that tertiary protein structure of lectin from Chinese Narcissus was similar to that of Narcissus pseudonarcissus,containing three functional domains specific for agglutination with the consensus sequence motif QXDXNXVXY involved in alpha-D-mannose recognition.Plant expression vectors were constructed and wild-type Arabidopsis plants transformed by the floral dip method,and transgenic plants were obtained. Compared with wild-type plants,the transgenic plants had no change in flowers,but lateral shoot was fewer,and its stem,leaf and pod were larger.